Thermoprecipitating polymer containing enzyme specific ligands, process for the preparation thereof, and use thereof for the separation of enzymes

ABSTRACT

The present invention provides novel thermoprecipitating polymers of formula 1 containing novel enzyme sensitive ligands of the formula 4, processes for the preparation thereof respectively, and to the use thereof for the separation of enzymes. In formula 1 R is hydrogen or methyl, 1 is an integer in the range of from 1 to 10, m and n are either 0 or 1, x and y are integers greater than 1.

FIELD OF THE INVENTION

The present invention relates to novel thermoprecipitating polymerscontaining enzyme specific ligands. The present invention also relatesto a process for the preparation of such enzyme specific ligands and tomonomers embodying such ligands. More particularly, the presentinvention also relates to a process for the separation of enzymes usingsuch thermoprecipitating polymers. The invention also relates to aprocess for the separation of lysozyme and lectins from a mixture ofproteins and lectins such as wheat germ agglutinin and tomatoagglutinin.

BACKGROUND OF THE INVENTION

The separation and recovery of biomolecules like enzymes andglycoproteins are critical cost determining steps in most of the downstream processes in biotechnology industries. Conventionally, separationof lysozyme from the crude sources has been done by salt precipitation(Hasegawa, Mineo, Yoshida, Kazuya, Miyauchi, Sakae, Terazono, Masami:U.S. Pat. No. 4,504,583, (1985)) or by ion exchange techniques(Hasegawa, Mineo, Ozaki, Kitao, U.S. Pat. No. 4,705,755 (1987); Durance,Timothy, Li-Chan, Eunice, Nakai, Shuryo: U.S. Pat. No. 4,966,851 (1990);Hasegawa, Mineo: U.S. Pat. No. 4,518,695 (1985); Takechi, Kaz,Takahashi, Tsuyos, Inaba, Toyoaki, Hasegawa, Eiichi: U.S. Pat. No.4,104,125 (1978).

Various techniques based on affinity interactions betweenenzyme-inhibitor, enzyme-substrate, enzyme-transition state analog,enzyme-cofactor and the like have been developed as better alternativesto the above-mentioned conventional systems for the selective recoveryof enzymes. Most of the affinity based separations involve polymers towhich the affinity ligand or cofactors or dyes are chemically linked.The complex formed between enzyme and the polymeric ligand issubsequently processed to isolate the enzyme.

These alternative systems primarily involve affinity chromatography oraffinity ultrafiltration. Although these techniques provide highselectivity, they are beset with several practical difficulties whichare summarised below.

Bozzano A G., Glatz C. E., J. Memb.Sci 55, 181-198 (1991); Ehsani N.,Parkkinen S., Nystrom M. J. Memb Sci 123, 105-119, (1997) disclose thataffinity ultrafiltration is suitable for the separation of enzyme onlyin cases where the difference between the molecular weights of thedesired enzyme and other biomolecules is appreciably large. Also, athigh pressures, the denaturation of the enzyme and fouling of themembrane leads to poor product quality (Ehsani N., Parkkinen S., NystromM. J. Memb Sci 123, 105-119, (1997)).

Affinity chromatography (Hirano S., Kaneko X, Kitagawa M., Agric. Biol.Chem, 55, 1683-1684 (1991); Safrik L., Safarikova M., J. BiochemBiophys. Methods, 27, 327-330 (1993); Reid, Lorne S.; U.S. Pat. No.4,552,845 (1985)) is found suitable only for small capacity columns.Scale up of the columns retards the flow rate and leads to dogging ofthe columns, thereby resulting in increased cost of the process. Anotherdisadvantage of this process is that it is non-continuous since periodicflushing is required at specified time intervals to remove anyundesirable non-specifically adsorbed biomolecules in the column (ChernC. S., Lee C. K., Chen C. Y., Yeh M. J., Colloids and Surfaces B.Biointerfaces. 6 37-49, (1996)).

Affinity precipitation eliminates many of the above-mentioned problemsand offers certain advantages such as ease of scale up, amenability tocontinuous operation and recycling of the affinity ligand (Chern C. S.,Lee C. K., Chen C. Y., Yeh M. J., Colloids and Surfaces B.Biointerfaces. 6 37-49, (1996)). Affinity precipitation involves theformation of a complex between the enzyme and a stimuli sensitiveaffinity polymer in a homogeneous solution. This complex is precipitatedby a change in the pH, temperature or ionic strength. This complex isdissociated and the polymer is separated by varying either one of theabove-mentioned stimuli and the enzyme is then isolated (Gupta M. N.,Kaul R., Guogiang D., and Mattiasson B., J. Mol. Recog. 9, 356-359,(1996)). Thus the recovery of the enzyme by this process is muchsimpler.

It is well known that ligands containing N-acetyl groups such asN-acetylglucosamine, N-acetylmuramic acid, chitin, chitosan, and thelike exhibit affinity for various enzymes and lectins (Tyagi R., KumarA, Sardar M., Kumar S., Gupta M. N., Isol.Purif. 2, 217-226, (1996);Katz, Friedrich D., Fishman, Louis, Levy; U.S. Pat. No. 3,940,317(1976)).

All the above ligands are derived from glucose. Since glucose is acarbons source for many microbes, such ligands are susceptible tomicrobial attack and hydrolytic degradation resulting in poor stabilityof these ligands (Hirano S., Kaneko H., Kitagawa M., Agric. Biol. Chem,55, 1683-1684 (1991)). Also chitosan is insoluble in alkaline mediawhile crosslinked chitosan and chitin are insoluble in both alkaline andacidic media (Ruckstein, Eli; Zeng, Xianfang; Biotechnology andBioengineering, 56, 610-617 (1997)). This limits their use. Moreover,chitin and chitosan can undergo transglycosylation and mutarotation,which drastically reduces their efficiency in any affinity basedrecovery of enzyme (Davies, R. C., Neuberger, A, Wilson, B. M., BiochemBiophys Acta, 178, 294-305, (1969); Neuberger, A, Wilson, B. M.,Biochem. Biophys. Acta, 147, 473-486, (1967)). Thus, it is desirable toreplace glucose by stable synthetic ligands during the synthesis ofthermoprecipitating affinity polymers for enzyme separations.

OBJECTS OF THE INVENTION

It is an object of the invention to provide thermoprecipitating polymersand processes for the preparation thereof, said thermoprecipitatingpolymers containing ligands having N-acetyl groups.

It is another object of the invention to provide stable syntheticligands for thermoprecipitating polymers for enzyme separation.

It is another object of the invention, to provide acrylic monomers andprocess for the preparation thereof which are useful in the synthesis ofthermoprecipitating polymers containing the above ligands havingN-acetyl groups.

It is a further object of the invention to provide an economical processfor the separation of lysozyme using the thermoprecipitating polymers ofthe invention.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 givers the graph of specific activity of the lysozyme recoveredin terms of unit activity/unit protein vs. number of cycles.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides thermoprecipitating polymersof the general formula 1

wherein R is hydrogen or methyl, 1 is an integer in the range of from 1to 10, m and n are either 0 or 1, x and y are integers greater than 1.

The present invention also relates to a process for the preparation ofthermoprecipitating polymers of the general formula 1

wherein R is hydrogen or methyl, 1 is an integer in the range of from 1to 10, m and n are either 0 or 1, x and y are both integers greater than1, said process comprising polymersing a thermosensitive monomer of thegeneral formula 3 with an affinity monomer of the general formula 2.

In one embodiment of the invention, the thermosensitive monomer (3) andthe affinity polymer (2) are polymerised by free radical polymerisation.

In another embodiment of the invention, the thermosensitive monomer usedis selected from the group consisting of N-isopropylacrylamide (NIPAM),N-isopropylmethacrylamide (NIPMAM), N-vinylcaprolactam (NVC) and vinylpyrrolidone (VP).

In a further embodiment of the invention, the molar ratio of theacrylated N-acetyl group containing ligand to the thermosensitivemonomer used is in the range of 1:1 to 1:9.

In yet another embodiment of the invention, the polymerisation initiatoris selected from the group consisting of azoisobutyronitrile (AIBN),ammonium persulfate (AMPS) and potassium persulfate.

In a further embodiment of the invention, the polymerisation initiatorused is in the range of 5 to 20% by weight of the monomers.

In yet another embodiment of the invention, the polymerisationaccelerator used is selected from the group consisting of N,N′,N″,N″Δtetramethylethylenediamine (TEMED) and sulfite containing compounds suchas sodium metabisulfite and potassium metasulfite.

In a further embodiment of the invention, the polymerisation acceleratorused is in the range of 1 to 5% by weight of the monomers.

The present invention also relates to novel monomers of the generalformula 2 embodying the ligand of formula 4

wherein R is hydrogen or methyl, 1 is an integer in the range of from 1to 10, m and n are either 0 or 1.

The invention also relates to a process for the preparation of anacrylated monomer of the general formula 2 comprising reacting a ligandcontaining N-acetyl groups of the general formula 4 with an acrylatemonomer in the presence of a condensing agent in a solvent at atemperature in the range of 0-25° C. for a period of 12 to 60 hours toobtain the acrylated monomer of formula 2.

In a further embodiment of the invention, the acrylic monomer isselected form compounds of the formula H₂C═CR—COCl andH₂C═CCH₃—CO—O—(CH₂)₂—OH wherein R is hydrogen or methyl.

In a further embodiment of the invention, the condensing agent isselected from the class of carbodiimide group compounds such asdicyclohexyl carbodiimide (DCC), 1-cyclohexyl3-(2-morphilinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

In a further embodiment of the invention, the molar ratio of the ligandcontaining N-acetyl group to the acrylate monomer is in the range of 1:1to 1:1.2.

In a further embodiment of the invention, the molar ratio of thecondensing agent to the acetamido ligand or acrylate monomer is in therange of 1:1 to 1:1.2.

The present invention also relates to novel ligands of the generalformula 4 below

wherein 1 is an integer in the range of from 1 to 10, m and n are either0 or 1.

The present invention also relates to a process for the preparation ofligands of the general formula 4 wherein 1 is an integer in the range offrom 1 to 10, m and n are either 0 or 1, said process comprisingreacting a straight chain amino acid of the general formula 5

with an acetylating agent in a solvent at a temperature in the range of0-25° C. for a period in the range of from 1-4 hours to obtain theligand of formula 4.

In one embodiment of the invention, the straight chain amino acid isselected from compounds of the formulaHOOC—(CH₂)₁—(CO—NH)_(m)—(CH₂)_(n)—NH₂ wherein 1 is an integer in therange of from 1 to 10, m and n are either 0 or 1.

In another embodiment of the invention, the solvent is selected from thegroup consisting of NaOH and water.

In a further embodiment of the invention, the acetylating agent isselected from acetic anhydride and acetyl chloride.

In yet another embodiment of the invention, the molar ratio of theacetylating agent to the amino acid is in the range of 1:1 to 1:1.2.

The invention also relates to a process. for the separation of lysozymecomprising polymerising acrylated monomer of the formula 2 with athermosensitive monomer of the formula 3 in the presence of apolymerisation initiator and polymerisation accelerator in a solvent at30 to 80° C. for 1 to 12 hours, to obtain the thermoprecipitatingaffinity polymer of formula 1, contacting the aqueous solution of thepolymer with an aqueous solution of lysozyme or a mixture of lysozymeand other proteins at a temperature in the range of 4 to 20° C. for atime period in the range of 1 to 16 hours followed by raising thetemperature above the LCST of the polymer, isolating the precipitatedpolymer-lysozyme complex, redissolving the said complex in an acidicaqueous solution, raising the temperature of the solution above the LCSTof the polymer, isolating the precipitated polymer and recoveringlysozyme from the solution.

DETAILED DESCRIPTION OF THE INVENTION

The N-acetyl group containing ligands are synthesised by dissolvingstraight chain amino acids of the formulaHOOC—(CH₂)₁—(CO—NH)_(m)—(CH₂)_(n)—NH₂ wherein 1 is an integer in therange of 1 to 10 and m and n are either 0 or 1 and equimolar NaOH inwater at 5-20° C., adding the acetylating agent dropwise to the abovesolution while maintaining, the pH in the range of 7.4-7.8 by theaddition of NaOH, stirring the reaction mixture for 2 hours at 25° C.,acidifying to pH 2.0, precipitating the product by cooling to 4° C. for24 hours, reprecipitating the product from water into acetone.

The monomers containing acrylated N-acetyl group are synthesised bydissolving the affinity lignad of the formulaHOOC—(CH₂)₁—(CO—NH)_(m)—(CH₂)_(n)—NH—CO—CH₃ wherein 1 is an integer inthe range of from 1 to 10 and m and n are either 0 or 1, and acrylatemonomer having the formula H₂C═CR—COCl or H₂C═CCH₃—CO—O—(CH₂)₂—OHwherein R is either hydrogen or methyl, and a condensing agent intetrahydrofuran, at 5-20° C., stirring this reaction mixture for 48hours at the same temperature, filtering the precipitate andconcentrating the filtrate to obtain the viscous liquid, pouring thisliquid into cold water to precipitate the product and then drying theproduct and preserving it under petroleum at 4° C.

The affinity precipitating polymers of the general formula 1 areprepared by dissolving the monomer containing acrylated N-acetyl groupof the formula 2 and thermosensitive monomer of the formula 3 in wateror dimethylformamide (DMF), purging this reaction mixture with nitrogenfor 10 to 30 minutes at 37 and 80° C. respectively, addingpolymerisation initiator and polymerisation accelerator, allowingpolymerisation to proceed for 5 to 8 hours under nitrogen purging at thesame temperature, precipitating the product either by increasing thetemperature of the aqueous solution above the LCST or in diethyl ether.

The, separation of lysozyme from other proteins is carried out using thethermoprecipitating polymer of the invention. An aqueous solution of thepolymer is mixed with lysozyme-ovalbumin mixture allowing equilibriumfor 8 to 24 hours, at 4 to 20° C., raising the solution temperatureabove the LCST (21 to 45° C.) of the affinity polymer therebyprecipitating the polymer-lysozyme complex, separating the precipitatedcomplex by centrifugation, redissolving the complex in an acidicsolution and again raising the temperature of the solution above theLCST of the affinity polymer, and then separating the filtratecontaining free lysozyme by centrifugation, repeating the procedure forsixteen cycles for determination of reusability and stability of thepolymers of the invention during recovery of lysozyme.

Although the present invention describes a process for the synthesis ofligands containing pendant N-acetyl groups and acrylated monomerscontaining N-acetyl groups and their subsequent use in the preparationof thermoprecipitating polymers useful in the enhanced recovery oflysozyme from a mixture of lysozyme and ovalbumin as well as reusabilityand enhanced stability of these polymers during recovery of lysozyme,the scope of the present invention is not and should not be construed tobe limited to only such affinity polymers for the separation of lysozymebut can extend to such combinations of polymer bound affinity ligandsand enzymes or lectins to which such ligands bind selectively.

The invention is also not limited to those ranges provided herein. Theranges and limitations provided herein are believed to particularlypoint out the invention. However, other ranges and limitations whichperform substantially the same function in the same or substantially thesame manner to obtain the same or substantially the same results areintended to be within the scope of the instant invention.

EXAMPLE 1

11.1 g (0.05 M) N-acetylglucosamine and 4.2 g (0.05M) sodium bicarbonatewas dissolved in 100 ml water. To this 5 ml (0.06M) acryloyl chloride in5 ml dichloromethane (DCM) was added dropwise over a period of 30minutes at 15° C. During this period the pH of the solution wasmaintained at 7.5 by adding saturated sodium bicarbonate solution. Afterthe addition of acryloyl chloride was over and the pH of the reactionmixture was steady, unreacted acryloyl chloride was extracted in 100 mlethyl acetate. The clear aqueous solution was separated and acidified topH 5.0 by concentrated HCl. Then the product (Ac.NAG) was previpitatedin acetone and maintained at 4° C. overnight to maximise theprecipitation. The precipitated product was filtered. The product wasreprecipitated from water into acetone. The thermoprecipitating polymercontaining this monomer was prepared as follows: 0.238 g (0.07M) NIPAM,0.248 g (0.03M) Ac.NAG were dissolved in 30 ml of water taken in a roundbottom flask. Polymerisation was initiated at 37° C. under nitrogenpurging by the addition of 10 (w/w) of AMPS and 1% v/w of TEMED.Polymerisation was allowed to proceed for 6 hours at 37° C. Then, thepolymer was precipitated by raising the temperature of the reactionmixture above the LCST of the polymer. The precipitated polymer waswashed twice with cold double distilled water and once with cold 0.066 Mphosphate buffer (pH 6.2) and dried under vacuum at room temperature.

EXAMPLE 2

21.3 g of acrylamide (0.3M), 0.5 g 4-(dimethylamino)-pyridine and 24.2ml (0.3M) pyridine was dissolved in 800 ml HPLC grade THF. The reactionmixture was stirred at 10° C. to obtain a clear solution. Then 21.3 ml(0.3M) acetyl chloride in 22 ml THF was added dropwise to the abovereaction mixture over 1 hour. After the addition of acetyl chloride wasover, a sticky yellow precipitate of pyridine-HCl was obtained. Thisprecipitate was filtered under vacuum to obtain a clear THF solution,which was then concentrated under vacuum at 40° C. to obtain a semisolidmass. This was precipitated in petroleum ether to get a low meltingsemisolid product (acetylacrylamide). The polymerisation comprising thismonomer was carried out as follows: 0.238 g (0.07M) NIPAM and 0.102 g(0.03M) acetylacrylamide were dissolved in 30 ml of water taken in around bottom flask. Polymerisation was initiated at 3 7° C. undernitrogen purging by the addition of 10 (w/w) of AMPS and 1% v/w ofTEMED. Polymerisation was allowed to proceed for 6 hours at 37° C. Then,the polymer was precipitated by raising the temperature of the reactionmixture above the LCST of the polymer. The precipitated polymer waswashed twice with cold double distilled water and once with cold 0.066Mphosphate buffer (pH 6.2) and dried under vacuum at room temperature.

EXAMPLE 3

7.5 g (0.1M) glycine and 4 g (0.1M) sodium hydroxide was dissolved in 80ml water. To it 9.5 ml (0.1M) acetic anhydride was added over a periodof 30 minutes at 15° C. During this addition, pH of the reaction mixturewas maintained at 7.5. After the addition was over, the reaction mixturewas stirred for two hours and acidified to pH 2.0 by concentrated HCl.It was maintained at −4° C. overnight. This reaction mixture was allowedto thaw and the precipitated product (2-acetamido glycine) was filtered.The product was reprecipitated from water into acetone. The monomer(2,(methacryloyl)oxy 2, ethylacetamidoglycinate) was prepared asfollows: in a round bottom flask, 4.1 g (0.035M) 2-acetamido glycine,4.3 ml (0.035M) 2, hydroxyethylmethacrylate (HEMA), 4.8 g (0.035M)1,hydroxybenzotriazole were dissolved in 300 ml dry THF. This reactionmixture was stirred at 15° C. under dry conditions to obtain a clearsolution. Then, 7.3 g (0.035M) DCC dissolved in 10 ml of THF was addedto the above reaction mixture. It was allowed to stir at 15° C. for 48hours. A heavy white precipitate of dicyclohexyl urea salt was filteredand the clear filtrate was concentrated under vacuum at 40° C. to obtainan oily semisolid mass. This was precipitated in cold water. The whiteproduct obtained was dried and preserved under hexane at 4° C. tillfurther use. Thermoprecipitating polymer was prepared as follows: 0.40 g(0.07M) NIPAM and 0.28 g (0.03M) 2,(methacryloyl)oxyethyl2,acetamidoglycinate were dissolved in 50 ml of dry DMF taken in a roundbottom flask. This was allowed to stir under continuous nitrogen purgingto obtain a clear solution. Then, the solution temperature was slowlyraised to 80° C. Polymerisation was initiated by adding 10% (w/w) ofAIBN dissolved in 1 ml DMF. This reaction was maintained at 80° C. for 6hours under continuous nitrogen purging The mass was cooled to 37° C.and the polymer was precipitated in diethyl ether. The precipitatedproduct was dried at 40° C.

EXAMPLE 4

8.9 g (0.1 M) β alanine and 4 g (0.1M) sodium hydroxide was dissolved in80 ml water. To it, 9.5 ml (0.1M) acetic anhydride was added over aperiod of 30 minutes at 15° C. During this addition pH of the reactionmixture was maintained at 7.5. After the addition was over, thisreaction mixture was stirred for two hours and acidified to pH 2.0 byconcentrated HCl. It was maintained at −4° C. overnight. This reactionmixture was allowed to thaw and the precipitated product (3-acetamidoβalaninate) was filtered. The product was reprecipitated from water intoacetone. The monomer 2,(methacryloyl)oxyethyl 3-acetamidoβalaninate) wasprepared as follows: in a round bottom flask 4.6 g (0.035M) 3-acetamidoβalaninate and 4.3 ml (0.035M) 1,hydroxybenzotriazole were dissolved in300 ml dry THF. This reaction mixture was stirred at 15° C. under dryconditions to obtain a clear solution. Then, 7.3 g (0.035M) DCCdissolved in 10 ml of THF was added to the above reaction mixture. Itwas allowed to stir at 15° C. for 48 hours. A heavy white precipitate ofdicyclohexyl urea salt was filtered and the clear filtrate wasconcentrated under vacuum at 40° C. to obtain an oily semisolid mass. Itwas dissolved in minimum amount of dry ethyl acetate and this clearsolution was precipitated in petroleum ether to obtain white solid,which was kept over petroleum ether till further use.Thermoprecipitating polymer was prepared as follows: 0.40 g (0.07M)NIPAM and 0.36 g (0.03M) 2,(methacryloyl)oxyethyl 2,acetamidoβalaninatewere dissolved in 50 ml of dry DMF taken in a round bottom flask Thiswas allowed to stir under continuous nitrogen purging to obtain a clearsolution. Then, the solution temperature was slowly raised to 80° C.Polymerisation was initiated by adding 10% (w/w) of AIBN dissolved in 1ml DMF. This reaction was maintained at 80° C. for 6 hours undercontinuous nitrogen purging, The mass was cooled to 37° C. and thepolymer was precipitated in diethyl ether. The precipitated product wasdried at 40° C.

EXAMPLE 5

10.3 g (0.1 M) 4 amino butyric acid and 4 g (0.1M) sodium hydroxide wasdissolved in 80 ml water. To it, 9.5 ml (0.1M) acetic anhydride wasadded over a period of 30 minutes at 15° C. During this addition pH ofthe reaction mixture was maintained at 7.5. After the addition was over,this reaction mixture was stirred for two hours and acidified to pH 2.0by concentrated HCl. It was maintained at −4° C. overnight. Thisreaction mixture was allowed to thaw and the precipitated product(4-acetamido butyric acid) was filtered. The product was reprecipitatedfrom water into acetone. The monomer 2,(methacryloyl)oxyethyl4-acetamidobutyrate) was prepared as follows: in a round bottom flask5.1 g (0.035M) 4-acetamido butyric acid and 4.3 ml (0.035M) HEMA and 4.8g (0.035M) 1, hydroxybenzotriazole were dissolved in 300 ml dry THF.This reaction mixture was stirred at 15° C. under dry conditions toobtain a clear solution. Then, 7.3 g (0.035M) DCC dissolved in 10 ml ofTHF was added to the above reaction mixture. It was allowed to stir at15° C. for 48 hours. A heavy white precipitate of dicyclohexyl urea saltwas filtered and the clear filtrate was concentrated under vacuum at 40°C. to obtain an oily semisolid mass. This was precipitated in coldwater. The white product obtained was dried and preserved under hexaneat 4° C. till further use. Thermoprecipitating polymer was prepared asfollows: 0.40 g (0.07M) NIPAM and 0.39 g (0.03M)2,(methacryloyl)oxyethyl 4-acetamidobutyrate were dissolved in 50 ml ofdry DMF taken in a round bottom flask. This was allowed to stir undercontinuous nitrogen purging to obtain a clear solution. Then, thesolution temperature was slowly raised to 80° C. Polymerisation wasinitiated by adding 10% (w/w) of AIBN dissolved in 1 ml DMF. Thisreaction was maintained at 80° C. for 6 hours under continuous nitrogenpurging. The mass was cooled to 37° C. and the polymer was precipitatedin diethyl ether. The precipitated product was dried at 40° C.

EXAMPLE 6

13.1 g (0.1 M) 6-amino caproic acid and 4 g (0.1M) sodium hydroxide wasdissolved in 80 ml water. To it, 9.5 ml (0.1M) acetic anhydride wasadded over a period of 30 minutes at 15° C. During this addition pH ofthe reaction mixture was maintained at 7.5. After the addition was over,this reaction mixture was stirred for two hours and acidified to pH 2.0by concentrated HCl. It was maintained at −4° C. overnight. Thisreaction mixture was allowed to thaw and the precipitated product(6-acetamidocaproic acid) was filtered. The product was reprecipitatedfrom water into acetone. The monomer 2,(methacryloyl)oxyethyl6-acetamidocaproate) was prepared as follows: in a round bottom flask6.1 g (0.035M) 6-acetamido caproic acid and 4.3 ml (0.035M) and 4.8 g(0.035M) 1, hydroxybenzotriazole were dissolved in 300 ml dry THF. Thisreaction mixture was stirred at 15° C. under dry conditions to obtain aclear solution. Then, 7.3 g (0.035M) DCC dissolved in 10 ml of THF wasadded to the above reaction mixture. It was allowed to stir at 15° C.for 48 hours. A heavy white precipitate of dicyclohexyl urea salt wasfiltered and the clear filtrate was concentrated under vacuum at 40° C.to obtain an oily semisolid mass. This was precipitated in cold water.The white product obtained was dried and preserved under hexane at 4° C.till further use. Thermoprecipitating polymer was prepared as follows:0.40 g (0.07M) NIPAM and 0.43 g (0.03M) 2,(methacryloyl)oxyethyl6-acetamidocaproate were dissolved in 50 ml of dry DMF taken in a roundbottom flask. This was allowed to stir under continuous nitrogen purgingto obtain a clear solution. Then, the solution temperature was slowlyraised to 80° C. Polymerisation was initiated by adding 10% (w/w) ofAIBN dissolved in 1 ml DMF. This reaction was maintained at 80° C. for 6hours under continuous nitrogen purging. The mass was cooled to 37° C.and the polymer was precipitated in diethyl ether. The precipitatedproduct was dried at 40° C.

EXAMPLE 7

13.2 g (0.1 M) glycylglycine and 8.4 g (0.1M) sodium bicarbonate wasdissolved in 80 ml water. To it, 9.5 ml (0.1M) acetic anhydride wasadded over a period of 30 minutes at 15° C. During this addition pH ofthe reaction mixture was maintained at 7.5 by the addition of saturatedsolution of lime. After the addition was over, this reaction mixture wasstirred for two hours and acidified to pH 2.0 by concentrated HCl. Itwas maintained at −4° C. overnight. This reaction mixture was allowed tothaw and the precipitated product (acetamido glycylglycinate) wasfiltered. The product was reprecipitated from water into acetone. Themonomer 2,(methacryloyl)oxyethyl acetamidoglycylglycinate) was preparedas follows: in a round bottom flask 6.1 g (0.035M) acetamidoglycylglycinate and 4.3 ml (0.035M) HEMA and 4.8 g (0.035M) 1,hydroxybenzotriazole were dissolved in 300 ml dry THF. This reactionmixture was stirred at 15° C. under dry conditions to obtain a clearsolution. Then, 7.3 g (0.035M) DCC dissolved in 10 ml of THF was addedto the above reaction mixture. It was allowed to stir at 15° C. for 48hours. A heavy white precipitate of dicyclohexyl urea salt was filteredand the clear filtrate was concentrated under vacuum at 40° C. to obtainan oily semisolid mass. This was precipitated in cold water. The whiteproduct obtained was dried and preserved under hexane at 4° C. tillfurther use. Thermoprecipitating polymer was prepared as follows: 0.40 g(0.07M) NIPAM and 0.43 g (0.03M) 2, (methacryloyl) oxyethyl acetamidoglycylglycinate were dissolved in 50 ml of dry DMF taken in a roundbottom flask. This was allowed to stir under continuous nitrogen purgingto obtain a clear solution. Then, the solution temperature was slowlyraised to 80° C. Polymerisation was initiated by adding 10% (w/w) ofAIBN dissolved in 1 ml DMF. This reaction was maintained at 80° C. for 6hours under continuous nitrogen purging. The mass was cooled to 37° C.and the polymer was precipitated in diethyl ether. The precipitatedproduct was dried at 40° C.

EXAMPLE 8

Relative binding of affinity thermoprecipitating polymer was determinedfollowing a procedure reported by Neuberger and Wilson, Biochem.Biophys. Acta 147, 473-486 (1967).

The concentration of N-acetyl groups required to obtain 35% relativebinding i.e. I₃₅ for various thermoprecipitating affinity polymers issummarized in Table 1. TABLE 1 Relative binding of thermoprecipitatingpolymers No. Affinity thermoprecipitating polymer mM N-acetyl groups forI₃₅ 1 Example 1 0.0050 ± 0.0004 2 Example 2 0.0160 ± 0.002  3 Example 30.0120 ± 0.001  4 Example 4 0.0120 ± 0.001  5 Example 5 0.0032 ± 0.00046 Example 6 0.0024 ± 0.0002 7 Example 7 0.0022 ± 0.0001

EXAMPLE 9

Ten millilitres of respective 1% w/v thermoprecipitating polymersolution was mixed with lysozyme and ovalbumin (27 μg/ml respectively)synthetic mixture. This solution was incubated at 20° C. for 16 hourswith continuous shaking at 200 rpm. Then, the temperature of the mixturewas raised above the LCST of the polymer to precipitate thelysozyme-polymer complex. The complex was separated by centrifugation at10000 rpm for 20 minutes above LCST. The precipitated polymer wasdissolved in dilute acetic acid solution (pH 2.0). The temperature ofthis dissociated complex was raised above LCST and the precipitatedpolymer was separated by centrifugation. The clear filtrate was assayedfor lysozyme using Micrococcus lysodeikticus as a substrate followingthe procedure reported by Neuberger and Wilson, Biochem. Biophys. Acta,147, 473-486, (1967). Lysozyme recovery was estimated in terms of unitactivity recovered/mM of N-acetyl groups present in the polymer and thedate is given in Table 2. TABLE 2 Recovery of lysozyme fromlysozyme-ovalbumin mixture Affinity thermo- Unit activity of lysozymerecovered/mM No. precipitating polymer N-acetyl groups from ovalbuminmixture 1 Example 1 17590 ± 200 2 Example 2 6352 ± 70 3 Example 3 6657 ±72 4 Example 4 7978 ± 65 5 Example 5 22516 ± 220 6 Example 6 16484 ± 1757 Example 7 33672 ± 320

EXAMPLE 10

Ten millilitres of respective 1% w/v thermoprecipitating polymersolution was mixed with 27 μg/ml lysozyme solution. It was allowed toincubate at 20° C. for 16 hours. Then, the temperature of the mixturewas raised above the LCST of the polymer to precipitate thelysozyme-polymer complex. The complex was separated by centrifugation at10000 rpm for 20 minutes above LCST. The precipitated polymer wasdissolved in dilute acetic acid solution (pH 2.0). The temperature ofthis dissociated complex was raised above LCST and the precipitatedpolymer was separated by centrifugation. The clear filtrate was assayedfor protein content using Lowrey's procedure and activity of lysozymewas measured using Micrococcus lysodeikticus as a substrate followingthe procedure reported by Neuberger and Wilson, Biochem. Biophys. Acta,147, 473-486, (1967). The graph of specific activity recovered in termsof unit activity/unit protein vs. number of cycles is given in FIG. 1.

The data in Table 2 shows that the activity of lysozyme revoered per mMof acetamido groups increased from 6657 units (see Example 3) to 33672units (see Example 7). Thus, in a lysozyme-ovalbumin mixturethermoprecipitating polymers exhibit selectivity towards lysozyme. Thepercent recovery in terms of specific activity (unit activity/unitprotein) as a function of the number of cycles is shown in FIG. 1. Thedata shows that even after 16 cycles the decrease in the percentrecovery in terms of specific activity was 21% and 20% in the case ofExample 6 and Example 7 respectively. But for Example 1, the decrease inthe percent recovery in terms of specific activity was 50%. Thus, thepolymers synthesised as reported in this invention are more stable ascompared to NAG containing polymer. Moreover, these polymers arereusable for 16 continuous cycles of solubility/precipitation.

Advantages of the Invention

-   -   1. The process of the. present invention enables synthesis of        thermoprecipitating affinity polymers exhibiting enhanced        interactions with the enzyme and thereby giving high recovery of        the desired enzyme.    -   2. The synthetic affinity polymers synthesized in the present        invention are reusable and stable during the recoveries of the        enzyme as compared to the N-acetylglucosamine containing        polymers.        References:        US Patents:

-   1. U.S. Pat. No. 4,504,583 Mineo et al

-   2. U.S. Pat. No. 4,705,755 Hasegawa et al

-   3. U.S. Pat. No. 4,966,851 Timothy et al

-   4. U.S. Pat. No. 4,518,695 Hasegawa et al

-   5. U.S. Pat. No. 4,104,125 Kaz et al

-   6. U.S. Pat. No. 4,552,845 Reid et al

-   7. U.S. Pat. No. 3,940,317 Katz et al    Publications

-   1. Bozzano A. G., Glatz C. E. J. Memb. Sci. 55 181-198 (1991)

-   2. Chem C. S., Lee C. K., Chen C. Y., Yeh M. J. Colloids and    Surfaces B. Biointerfaces, 637-49 (1996)

-   3. Davies R. C., Neuberger A., Wilson B. M., Biochim. Biophys. Acta,    178 294-305 (1969)

-   4. Ehsani N., Parkkinen S., Nystrom M., J. Memb. Sci. 123, 105-119,    (1997)

-   5. Gupta M. N., Kaul R., Guogiang D., Mattiasson B., J. Mol. Recog.,    9, 356-359, (1996)

-   6. IIirano S;, Kancko H., Kitagawa M., Agric. Biol. Chem 55,    1683-1684, (1991)

-   7. Kobayashi, Kazukiyo; Kamiya, Shoko; Matsuyama, Minoru; Murata,    Takeomi; Usui, Taichi, Polymer Journal, 30, 653-658 (1998)

-   8. Neuberger A., Wilson B. M., Biochim. Biophys. Acta., 147, 473-486    (1967)

-   9. Ruckenstein, Eli; Zeng, Xianfang, Biotechnology and    Bioengineering, 56, 610-617, (1997)

-   10. Safrik I., Safarikova M., J. Biochem. Biophys. Methods, 27,    327-330, (1993)

-   11. Tyagi R., Kumar A., Sardar M., Kumar S., Gupta M. N., Isol.    Purif, 2, 217-226, (1996)

1-22. (canceled)
 23. A monomer selected from the formulas

wherein R is hydrogen or methyl, 1 is an integer from 1 to 10, m is 0 or1 and n is 0 or
 1. 24. A process for preparing an acrylated monomerselected from a compound of the formulas

wherein R is hydrogen or methyl, 1 is an integer from 1 to 10, m is 0 or1 and n is 0 or
 1. comprising the steps of a) reacting a ligandcontaining of Formula 4

wherein 1 is an integer of from 1 to 10, m is 0 or 1 and n is 0 or 1with an acrylate monomer in the presence of a condensing agent in asolvent at a temperature in the range of 0-25° C. for a period of 12 to60 hours to obtain the acrylated monomer of formula (2a), (2b), or (2c).25. The process as claimed in claim 24, wherein acrylate monomer isselected from the group consisting of a H₂C═CR—COCl andH₂C═CCH₃═CO—O—(CH₂)₂—OH wherein R is hydrogen or methyl.
 26. The processas claimed in claim 24, wherein the condensing agent is a carbodiimidegroup
 27. The process according to claim 26, wherein the carbodiimide isselected from the group consisting of dicyclohexyl carbodiimide (DCC),1-cyclohexyl 3-(2-morphilinoethyl)carbodiimide metho-p-toluenesulfonate(CMC) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
 28. Theprocess as claimed in claim 24, wherein the molar ratio of the ligandcontaining N-acetyl group to the acrylate monomer is in the range of 1:1to 1:1.2.
 29. The process as claimed in claim 24, wherein the molarratio of the condensing agent to the ligand or acrylate monomer is inthe range of 1:1 to 1:1.2.
 30. A compound of Formula 4

wherein 1 is an integer of from 1 to 10, m is 0 or 1 and n is 0 or 1.31. A process for the preparation of a ligand of Formula 4

wherein 1 is an integer of from 1 to 10, m is 0 or 1 comprising thesteps of reacting a straight chain amino acid of Formula 5

where 1, m and n are as defined above with an acetylating agent in asolvent at a temperature in the range of 0-25° C. for a period in therange of from 1-4 hours to obtain the ligand of Formula
 4. 32. Theprocess as claimed in claim 31, wherein the solvent is selected from thegroup consisting of NaOH and water.
 33. The process as claimed in claim31, wherein the acetylating agent is selected from the group consistingof acetic anhydride and acetyl chloride.
 34. A process as claimed inclaim 31, wherein the molar ratio of the acetylating agent to the aminoacid of formula 5 is in the range of 1:1 to 1:1.2.